Standard methylation analysis methods provide only qualitative or semi-quantitative data, which can lead to inaccurate conclusions regarding the effects of epigenetic DNA methylation on cell cycle and metabolism. Without quantification of methylation levels, it is not possible to distinguish physiologically relevant methylation from background methylation.

Pyrosequencing solves this limitation by generating highly reproducible quantification of methylation frequencies at individual consecutive CpG sites (see figures Pyrosequencing analysis of CpG methylation pattern in the RASSF1A gene and Linearity of methylation quantification by Pyrosequencing). As such, Pyrosequencing can detect and quantify even small changes in methylation levels. Other valuable features include the inherent quality control afforded by the sequence context of results and the ability to compare results to expected methylation levels. Built-in controls for the bisulfite treatment eliminate manual estimation of non-converted DNA levels and prevent false-positive methylation detection, thereby ensuring the reliability of results.

These features have established Pyrosequencing as the gold standard for DNA methylation analysis. This technology has been used to correlate DNA methylation to tumor type and gene expression, to measure cellular response to treatment with demethylating agents, and to assess changes in methylation state in relation to tumorigenesis, genetic imprinting, and exposure to environmental toxins (1).

To characterize the methylation status of a DNA sequence via Pyrosequencing, the DNA is first incubated with sodium bisulfite. As a result, unmethylated cytosine residues are converted into uracil while methylated cytosines remain unchanged, giving rise to two different sequences that can be distinguished. An internal control for bisulfite treatment is incorporated into analysis. Cytosines that are not followed by guanine in template sequences are not methylated, and should therefore be converted to thymine by bisulfite treatment and PCR (see table Sequences resulting from bisulfite conversion and PCR). Full bisulfite conversion is confirmed if all templates show thymine and no cytosine in these positions. With unique DNA Protect technology, QIAGEN EpiTect Bisulfite Kits facilitate complete conversion and minimal degradation of the treated DNA.

The analysis of methylation status exploits the quantitative nature of Pyrosequencing data. Unlike Sanger sequencing, the peak heights in the resulting Pyrogram report the ratio of cytosine to thymine at each analyzed CpG site, which reflects the proportion of methylated DNA. Assay design is flexible — it can be performed in forward or reverse orientations, on either the top or the bottom strands. In addition, contiguous CpG sites are analyzed independently and within the same run, which enables assessment of sequence-wide methylation patterns while retaining details of position specific methylation (see figure Analysis of 16 CpG sites in a long sequence run).

The new CpN mode of the PyroMark Q24 Advanced system now enables methylation analysis of cytosine residues that are not part of CpG sites. Analysis of CpN and CpG sites can be performed together in a single Pyrosequencing reaction. Each of these positions can be selected individually in the software during the run setup. This example shows the analysis of a sequence with a CpN site (CpA in this case) in the first position, followed by 2 classical CpG sites (see figure New mode for analyzing CpN methylation).

Previously, analysis of methylation sites further away from the sequencing primer could be uncertain, but now with longer read lengths and higher accuracy of the PyroMark Q24 Advanced system, methylation quantification is highly reliable throughout the entire sequencing run. Bisulfite conversion in DNA methylation analysis leads to frequent poly T stretches in the nucleotide sequence, and analysis of CpG sites directly after such T homopolymers has previously been challenging due to uncertain quantification of the light signal at these sites. The increased accuracy of PyroMark Q24 Advanced enables reliable quantification of CpG methylation behind and even within a stretch of 8 T nucleotides (see figure Methylation quantification in homopolymers).